Expression of foreign genes using a replicating polyprotein producing virus vector

ABSTRACT

The present invention provides an expression vector adapted for expressing heterologous proteins in plants susceptible to a polyprotein-producing plant virus. The vector utilizes the unique ability of viral polyprotein proteases to cleave heterologous proteins from viral polyproteins. Also provided is a method for expressing heterologous proteins in plants using these unique expression vectors.

This invention was made with government support under Grant No. AI27832,awarded by the National Institutes of Health, Grant No. 91-37303-6435,awarded by the U.S. Dept. of Agriculture, and Grant No. IBN-9158559,awarded by the National Science Foundation. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to polyprotein-producing viral vectors forproduction of non-native proteins and which employ proteolytic digestionof the non-native proteins by the viral vector's own proteases.

2. Description of the Prior Art

Recombinant DNA techniques and genetic technologies have led to progressin the development of gene transfer into organisms, both plant andanimal. Investigators have attempted to produce pharmaceuticals,chemicals, and biologicals by gene transfer techniques. Even though agene may have been identified, cloned, and engineered, it is stillnecessary to introduce the gene into a host cell in which the gene maybe expressed.

Foreign genes may be expressed in the host from DNA integrated into thehost genome (transgenic organism), or from extrachromosomal pieces ofDNA or RNA which are not inherited (through transient expressionvectors). A variety of methods have been developed and are currentlyavailable for the transformation of various cells with genetic material.The most success has been achieved with dicotyledonous plants with somelimited success reported for the monocotyledonous plants. The mosthighly developed system for higher plants transformation is derived fromthe tumor-inducing mechanism of the soil bacterium Agrobacteriumtumefaciens. The Agrobacterium/Ti plasmid system exploits the elementsof the Agrobacterium transformation mechanism. Alternative methods fordelivering genetic material to plants include the direct incorporationof DNA such as the transformation of plant cell protoplast. Othertechniques include microinjection or DNA bombardment.

Transient expression vectors can be introduced directly into therecipient cell, and result in gene expression in a short period of time,generally 24-48 hours. Viral vectors (both DNA and RNA) offerpossibilities as plant and animal transformation vectors. Viral vectorshave some advantages over vectors involving non-viral DNA in certaincircumstances. Advantages of viral vectors include improvements in theease of gene introduction.

Viruses that have been shown to be useful for the transformation ofplant hosts include caulimovirus (CaMV), tobacco mosaic virus (TMV) andbrome mosaic virus (BMV). Transformation of plants using plant virusesis described in U.S. Pat. No. 4,855,237 (bean golden mosaic virus), EP-A67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA194,809 (BMV), EPA 278,667 (BV), Brisson, N. et al., Met. Enzymol.118:659 (1986) (CaMV), Guzman et al. Commun. Molec. Biol.: ViralVectors, Cold Spring Harbor Laboratory, New York, pp. 172- 189 (1988),and WO 93/03161 (TMV). Pseudovirus particles for use in expressingforeign DNA in many hosts, including plants, is described in WO87/06261.

For RNA viruses, a primary consideration is designing a vector thatproduces infectious transcripts, and therefore preserves the terminalstructure of the RNA in order for the transcript to be a good substratefor the viral replicase. Bacteriophage promoters have proven ideal fordirecting the synthesis of RNA from DNA clones. One RNA virus expressionvector, pPM1, utilized a modified lambda phage P_(r) promoter. Thisvector was used to synthesize infectious transcripts of brome mosaicvirus (BMV) genomic RNA from BMV cDNA clones. However, most of theseviral vectors have not been capable of systemic spread in the plant andexpression of the non-viral foreign genes in the majority of the plantcells in the whole plant. Another disadvantage of many of the prior artviral vectors is that they are not stable for the maintenance ofnon-viral foreign genes. The use of RNA vectors has also been limited bythe instability of inserted foreign sequences and disruption of systemicvirus spread after replacement of virus genes involved in movement. TMVviral vectors require a subgenomic promoter for operation. The prior artviral vectors do not provide a means for using viral proteases to cleavethe foreign proteins made by the transformed plants. Consequently, thereis a need for vectors which enable enhanced systemic expression of anucleic acid sequence after a short inoculation time and for vectorswhich can be used to produce both native and non-native proteinsrelative to the host cell, particularly plant cells, while allowingcleavage of the produced protein by viral proteases. cl SUMMARY OF THEINVENTION

It is an object of the invention to provide a polyprotein-producingvirus expression vector adapted to encoding at least one sequence for atleast one heterologous protein. The vector may be used to expressheterologous proteins in plants or plant cells.

Accordingly, the present invention provides a viral expression vectorencoding for at least one protein non-native to the vector that isreleased from at least one polyprotein expressed by said vector byproteolytic processing catalyzed by at least one protease in saidpolyprotein, said vector comprising: (a) at least one promoter; (b)cDNA, wherein said cDNA comprises a cDNA sequence which codes for atleast one polyprotein from a polyprotein-producing virus; (c) at leastone unique restriction site flanking a 3' terminus of said cDNA; and (d)a cloning vehicle.

The present invention also provides a viral expression vector encodingfor at least one protein non-native to the vector that is released fromat least one polyprotein expressed by said vector by proteolyticprocessing catalyzed by at least one protease in said polyprotein, saidvector comprising: (a) at least one promoter; (b) cDNA, wherein saidcDNA comprises a cDNA sequence which codes for at least one polyproteinfrom a polyprotein-producing virus, and wherein said polyproteincomprises at least one protein non-native to the vector; (c) at leastone unique restriction site flanking a 3' terminus of said cDNA; and (d)a cloning vehicle.

The present invention also provides a method for producing an expressionvector encoding for at least one selected protein non-native to thevector that is released from at least one polyprotein expressed by saidvector by proteolytic processing catalyzed by proteases in saidpolyprotein, said method comprising: (a) reverse transcribing apolyprotein-producing RNA into cDNA; (b) introducing at least one uniquerestriction site flanking a 3' terminus of said cDNA; and (c) insertingsaid cDNA into a cloning vehicle.

The present invention also provides a method for producing an expressionvector, adapted for expressing in a host cell at least one selectedprotein non-native to the vector that can be released from at least onepolyprotein by proteolytic processing catalyzed by proteases in saidpolyprotein, said method comprising: (a) reverse transcribing apolyprotein-producing RNA into a first cDNA; (b) introducing at leastone unique restriction site flanking a 3' terminus of said first cDNA;and (c) inserting into said first cDNA a second cDNA sequence whereinsaid second cDNA sequence encodes a protein non-native to the vector;and (d) inserting said first and second cDNA into a cloning vehicle.

The present invention also provides a method for expressing at least oneprotein in a host cell, wherein the protein is non-native to the hostcell, said method comprising infecting a host cell susceptible to apolyprotein-producing virus with an expression vector, wherein saidvector comprises a cDNA sequence encoding at least one polyprotein, eachof said polyprotein comprising at least one protein non-native to thevector and at least one protease, and wherein said protein non-native tothe vector is released from said polyprotein by proteolytic processingcatalyzed by said protease encoded by said cDNA, and expressing saidprotein non-native to the vector in said host cell.

These and other advantages of the present invention will become apparentfrom the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

"The file of this patent contains at least one drawings executed incolor. Copies of this patent with color drawing(s) will be provided bythe Patent and Trademark Office upon request and payment of thenecessary fee."

FIG. 1 depicts diagrams of portions of plasmids containing cDNArepresenting the complete TEV genome and inserted GUS gene.

FIG. 2 depicts immunoblot analysis of extracts from RNAtranscript-inoculated tobacco plants.

FIG. 3 depicts immunoblot analysis of extracts from tobacco plantsinfected by serially passaged TEV-GUS.

FIG. 4A depicts analysis of GUS-HC-Pro fusion protein and FIG. 4B showsGUS activity in aging plants infected by TEV-GUS.

FIG. 5 depicts in situ localization of GUS activity in plants infectedby TEV-GUS.

FIG. 6 depicts in situ localization of GUS activity in cross-sections ofpetioles, stems, and roots of plants infected by TEV-GUS.

FIGS. 7A-7B depict genetic maps of TEV and selected TEV variants used inthis study. FIG. 7(A) shows wild-type TEV-HAT as derived fromtranscription of pTEV7DA. FIG. 7(B) depicts TEV-GUS and deletionvariants.

FIG. 8 depicts immunoblot analysis of extracts from upper leaves ofTEV-GUS-inoculated tobacco plants using anti-HC-Pro sera.

FIG. 9 depicts relative positions of deletion endpoints within the GUSand HC-Pro coding sequences in spontaneous mutants of TEV-GUS.

FIGS. 10A-10B depict immunoblot analysis with anti-HC-Pro serum ofextracts from plants inoculated with synthetic RNA transcripts.

FIGS. 11A, B, C and D depict quantitation of TEV capsid protein (A, B)and RNA (C, D) present in systemically infected leaves of plantsinoculated with wild-type and mutant viruses.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to viral vector expressionsystems for inserting selected genes into host cells. The presentinvention facilitates the production of selected proteins using apolyprotein-producing virus vector. As used herein, the term"polyprotein-producing virus" means a virus from the picorna-likesupergroup as, for example, those described in Dolja, V. V. andCarrington, J. C., Seminars in Virology 3:315-326 (1992), incorporatedherein by reference, which produces a polyprotein. The picorna-likesupergroup of viruses encompass both plant and animal viruses.Picorna-like plant viruses are the most preferred. "Polyprotein" means aprotein encoded by a virus from the picorna-like supergroup that can bepost-translationally cleaved by one or more proteases.

Polyprotein-producing plant viruses include, but are not limited to,potyviruses, nepoviruses, and comoviruses. Potyviruses useful in thepresent invention include, but are not limited to, tobacco etchpotyvirus (TEV), and the viruses referenced in Edwardson, J. R. andChristie, R. G., "The Potyvirus Group: Monograph No. 16 (Agric. Exp.Station, Univ. of Florida, 1991), incorporated by reference herein.Animal viruses from the picorna-like supergroup that producepolyproteins include, but are not limited to picorna viruses and polioviruses.

As used herein the term "vector" refers to a vector produced with apolyprotein-producing plant virus cDNA and a cloning vehicle. In apreferred embodiment, the vector includes a coding sequence non-nativeto the viral vector inserted between coding sequences of the viralgenome. Vectors useful in the present invention generate a non-nativeprotein relative to the virus in the host as part of a polyprotein.Native viral proteases present in the vector cleave the polyprotein intoindividual proteins. "Protein non-native to the viral vector" or"non-native protein"as used herein means a protein(s) or polypeptide(s)that is not expressed by the wild-type virus. A promoter is a DNAsequence that binds RNA polymerase and facilitates initiation oftranscription.

In a preferred embodiment, the polyprotein-producing virus is TEV. TEVis awell-characterized potyvirus, and contains a positive-strand RNAgenome of 9.5 kilobases (kb) coding for a single, large polyprotein thatis processed by three virus-specific proteinases. The nuclear inclusionprotein "a" proteinase is involved in the maturation of severalreplication-associated proteins and capsid protein. The helpercomponent-proteinase (HC-Pro) and 35-kDa proteinase both catalyzecleavageonly at their respective C-termini. The proteolytic domain ineach of theseproteins is located near the C-terminus. The 35-kDaproteinase and HC-Pro derive from the N-terminal region of the TEVpolyprotein.

The different polyprotein-producing viruses have different proteinases,butall are considered to cleave both native and non-native proteins fromthe respective polyproteins.

The present invention demonstrates that polyprotein-producing virusvectorsin general, preferably potyvirus vectors, and most preferably TEVvectors, serve as efficient expression vectors. The theoretical yield ofprotein products encoded by a polyprotein-producing vector is extremelyhigh because all virus genome-encoded proteins are synthesized inequimolar amounts.

Introduction of the vector into the host results in production of thenon-native protein contained on the vector during the course of systemicinfection of the recombinant virus. The gene encoding the non-nativeprotein is introduced into the virus genome such that the non-nativeprotein is produced as part of a polyprotein precursor with other nativeviral proteins. Preferably, the sequence encoding the non-native proteinis inserted between coding sequences for native proteins of the viralgenome. The sequence encoding the non-native protein must have flankingcleavage sites to facilitate removal of the non-native protein from thepolyprotein. The engineering of site-specific cleavage sites recognizedbythe viral proteinases permits proteolytic release of the non-nativeproteinfrom the polyprotein. Techniques commonly used in the art toeffect site directed mutagenesis may be used. These include, forexample, the methods described by Kunkel, T. A., et al., MethodsEnzymol., 154:367-382 (1982).

In the case where a plant is used as the host cell, the vector shouldpreferably be aphid transmission-defective to prevent transmission ofthe recombinant vector to other plants. This is accomplished byinactivation of viral components necessary for insect transmissibility,such as, for example, the HC-Pro proteinase in TEV. Introduction ofmutations, such as deletions at sites in the insect-transmissibledependent proteins leads totransmission-defective vectors. Mutations maybe generated by techniques known to those skilled in the art.

The method for generating a vector comprises reverse transcription of apolyprotein-producing viral RNA by any reverse transcription methodcommonly used in the art to produce a cDNA. The cDNA is preferably fulllength, but partial cDNA may also be used, as long as the cDNA is ableto code for a functional polyprotein. Double-stranded DNA (d.s. DNA) isgenerated from the cDNA using any technique known to those skilled inthe art, such as, but not limited to, bacterial DNA polymerase and RNaseH. Unique restriction endonuclease sites may be added. The d.s. DNA isdigested with a suitable restriction endonuclease and the digest isinserted into a cloning vehicle (which may have at least one uniquerestriction endonuclease site) such as, but not limited to, a plasmid,cosmid or lambda. Resulting expression vectors are propagated in hostcells using techniques known to those skilled in the art.

Unique restriction sites are added to the gene encoding the non-nativeprotein to be inserted into the vector. These restriction sites may codefor cleavage at the N- or C-termini of the non-native protein (i.e., atthe 5' or 3' end). In one embodiment, restriction sites are generatedfor cleavage by the 35K (P1) protease to maturate the protein. In analternative embodiment, sites for cleavage at the C-terminus by NIaprotease may be introduced.

The gene coding for a protein non-native to the viral vector is insertedinto a specific site on the resulting cloning vehicle using anytechnique known to those skilled in the art for introducing foreigngenes into cloning vehicles. The transcripts may be applied to recipientcells by anytechnique known to those skilled in the art. These include,but are not limited to, manual application, such as abrasiveinoculation, particle bombardment, and/or electroporation.

As used herein, the phrase, "recipient cell" or "host cell" is intendedto refer to cells that are susceptible to infection by apolyprotein-producing virus. Species from, for example, the Solanaceae,Chenopodiaceae, Leguminosae, Amaranthaceae, Compositae, Campanulaceae,Scrophulariaceae, Convolvulaceae, Lobeliaceae families and others whichare known to be infected by potyvirus may be transformed. For example,Table 1 shows the species and Families which are known to be infected byTEV as described by Edwardson, J. R., and Christie, R. G., previouslyincorporated by reference. Additional species may be found which arealso susceptible to TEV infection.

                  TABLE 1                                                         ______________________________________                                        Species Reported to be Infected by Tobacco Etch Virus                         Species              Families                                                 ______________________________________                                        Alonsoa linearis     Scrophulariaceae                                         Amaranthus caudatus  Amaranthaceae                                            Atriplex hortensis   Chenopodiaceae                                           Beta vulgaris        Chenopodiaceae                                           Brachycome iberidifolia                                                                            Compositae                                               Browallia major      Solanaceae                                               B. speciosa          Solanaceae                                               Callistephus chinensis                                                                             Compositae                                               Campanula drabifolia Campanulaceae                                            Capsicum annuum      Solanaceae                                               C. frutescens        Solanaceae                                               C. microcarpum       Solanaceae                                               C. pendulum          Solanaceae                                               Cassia occidentalis  Leguminosae                                              C. tora              Leguminosae                                              Celosia argentia     Amaranthaceae                                            Charieis heterophylla                                                                              Compositae                                               Chenopodium album    Chenopodiaceae                                           C. amaranticolor     Chenopodiaceae                                           C. foetidum          Chenopodiaceae                                           C. quinoa            Chenopodiaceae                                           Cirsium vulgare      Compositae                                               Collinsia bicolor    Scrophulariaceae                                         C. heterophylla      Scrophulariaceae                                         Cuscuta californica  Convolvulaceae                                           C. lupulifornis      Convolvulaceae                                           Cymbalaria muralis   Scrophulariaceae                                         Datura ferox         Solanaceae                                               D. metel             Solanaceae                                               D. meteloides        Solanaceae                                               D. stramonium        Solanaceae                                               Dimorphotheca aurantiaca                                                                           Compositae                                               D. pluvialis         Compositae                                               D. sinuata           Compositae                                               Emmenanthe penduliflora                                                                            Hydrophyllaceae                                          Eupatorium lasseauxii                                                                              Compositae                                               Gamolepis tagetes    Compositae                                               Gomphrena globosa    Amaranthaceae                                            Gypsophila elegans   Caryophyllaceae                                          Helianthus annuus    Compositae                                               Helipterum humboldtianum                                                                           Compositae                                               Hyoscyamus niger     Solanaceae                                               Indigofera hirsuta   Leguminosae                                              Lamium amplexicaule  Labiatae                                                 Linaria bipartita    Scrophulariaceae                                         L. canadensis        Scrophulariaceae                                         L. maroccana         Scrophulariaceae                                         Lobelia erinus       Lobeliaceae                                              L. gracilis          Lobeliaceae                                              L. hederacea         Lobeliaceae                                              L. inflata           Lobeliaceae                                              L. tenuior           Lobeliaceae                                              Lycium chinensis     Solanaceae                                               Lycopersicon esculentum                                                                            Solanaceae                                               L. hirsutum          Solanaceae                                               L. peruvianum        Solanaceae                                               L. pimpinellifolium  Solanaceae                                               Margaranthus solanaceus                                                                            Solanaceae                                               Melilotus albus      Leguminosae                                              M. indicus           Leguminosae                                              M. italicus          Leguminosae                                              M. messanensis       Leguminosae                                              M. officinalis       Leguminosae                                              M. sulcatus          Leguminosae                                              M. wolgicus          Leguminosae                                              Mollugo verticillata Aizoaceae                                                Nemophilia insignis  Hydrophyllaceae                                          N. maculata          Hydrophyllaceae                                          N. merziesii         Hydrophyllaceae                                          Nicandra physaloides Solanaceae                                               Nicotiana acuminata  Solanaceae                                               N. alata             Solanaceae                                               N. benthamiana       Solanaceae                                               N. bigelovii         Solanaceae                                               N. bonariensis       Solanaceae                                               N. caudigera         Solanaceae                                               N. clevelandii       Solanaceae                                               N. digluta           Solanaceae                                               N. x edwardsonii     Solanaceae                                               N. glauca            Solanaceae                                               N. glutinosa         Solanaceae                                               N. langsdorfii       Solanaceae                                               N. longiflora        Solanaceae                                               N. nudicaulis        Solanaceae                                               N. paniculata        Solanaceae                                               N. pauciflora        Solanaceae                                               N. plumbaginifolia   Solanaceae                                               N. raimondii         Solanaceae                                               N. repanda           Solanaceae                                               N. rustica           Solanaceae                                               N. sanderae          Solanaceae                                               N. solanifolia       Solanaceae                                               N. sylvestris        Solanaceae                                               N. tabacum           Solanaceae                                               N. tomentosa         Solanaceae                                               N. trigonophylla     Solanaceae                                               N. undulata          Solanaceae                                               Niermbergia hippomanica                                                                            Solanaceae                                               Nolana lanceolata    Solanaceae                                               Penstemon grandiflorus                                                                             Scrophulariaceae                                         Petunia hybrida      Solanaceae                                               P. violaceae         Solanaceae                                               Phacelia campanularia                                                                              Hydrophyllaceae                                          P. ciliata           Hydrophyllaceae                                          P. grandiflora       Hydrophyllaceae                                          P. viscida           Hydrophyllaceae                                          P. whitlavia         Hydrophyllaceae                                          Physalis alkekengi   Solanaceae                                               P. angulata          Solanaceae                                               P. ciliosa           Solanaceae                                               P. floridana         Solanaceae                                               P. heterophylla      Solanaceae                                               P. ixocarpa          Solanaceae                                               P. peruviana         Solanaceae                                               P. pruinosa          Solanaceae                                               P. pubescens         Solanaceae                                               P. subglabrata       Solanaceae                                               Plantago lanceolata  Plantaginaceae                                           Portulaca oleracea   Portulacaceae                                            Primula malacoides   Primulaceae                                              Proboscidea jussieui Martynaceae                                              Rudbekia amplexicaulis                                                                             Compositae                                               Salpiglosis sinuata  Solanaceae                                               Schizanthus pinnatus Solanaceae                                               Senecio vulgaris     Compositae                                               Silene angelica      Caryophyllaceae                                          Solanum aculeatissimum                                                                             Solanaceae                                               S. capsicastrum      Solanaceae                                               S. carolinense       Solanaceae                                               S. elaegnifolium     Solanaceae                                               S. integrifolium     Solanaceae                                               S. melongena         Solanaceae                                               S. nigrum            Solanaceae                                               S. pseudocapsicum    Solanaceae                                               S. seaforthianum     Solanaceae                                               S. tuberosum         Solanaceae                                               Spinacia oleracea    Chenopodiaceae                                           Tetragonia expansa   Aizoaceae                                                Torenia fournieri    Scrophulariaceae                                         Trigonella calliceras                                                                              Leguminosae                                              T. coerulea          leguminosae                                              T. corniculata       Leguminosae                                              T. cretica           Leguminosae                                              T. foenum-graecum    Leguminosae                                              Valerianella locusta Valeranaceae                                             Verbena canadensis   Verbenaceae                                              V. hybrida           Verbenaceae                                              Zaluzianskya villosa Scrophulariaceae                                         Zinnia elegans       Compositae                                               ______________________________________                                    

Host cells may be in plants, plant cell cultures, animals or animal cellcultures. Cell cultures include any conventional in vitro cell culturesuch as, but not limited to, roller bottle, agar, and bioreactortechniques. The host cell may be a plant or an animal cell. It isanticipated that vectors having plant polyprotein-producing viral RNAwillinfect susceptible plant cells, and that vectors having animalpolyprotein-producing viral RNA will infect susceptible animal cells,although some plant host cells may be susceptible to animalpolyprotein-producing viral RNA, and some animal host cells may besusceptible to plant polyprotein-producing viral RNA. "Cell" as usedherein may be a single cell, a plurality of cells, or an organism.

In order to improve the ability to identify transformants, one maydesire to employ selectable or screenable marker gene as, or in additionto, the expressible gene of interest. Such genes may encode either aselectable orscreenable marker, depending on whether the marker confersa trait which one can select for by chemical means, i.e., through theuse of a selectiveagent such as an herbicide, antibiotic or the like, orwhether it is simplya trait that one can identify through observation ortesting (e.g., the R-locus trait). Many examples of suitable markergenes are known to the art and can be employed in the practice of thepresent invention.

Possible selectable markers for use in connection with the presentinvention include, but are not limited to, a neo gene which codes forkanamycin resistance and can be selected for using kanamycin, G418,etc.; a bar gene which codes for bialaphos resistance, a mutant EPSPsynthase gene which encodes glyphosate resistance; etc. Exemplaryscreenable markers include beta-glucuronidase (GUS), or an R-locus gene,which encodes a product that regulates the production of anthocyaninpigments (red color) in host cells. Included within the terms"selectable" or "screenable marker" genes are also genes which encode asecretable marker whose secretion can be detected as a means ofidentifying or selecting fortransformed host cells. Examples includemarkers which are able to secrete antigen(s) that can be identified byantibody interaction, or an enzyme(s)which can be detectedcatalytically.

The choice of the particular foreign DNA segments to be delivered to therecipient host cells will often depend on the purpose of thetransformation.

The vector is used to generate proteins of interest that are not nativeto the virus in a host cell. This includes the production of importantproteins or other products for commercial use, such as lipase, melanin,pigments, antibodies, hormones, pharmaceuticals such as, but not limitedto, interleukins, EPO, G-CSF, GM-CSF, hPG-CSF, M-CSF, Factor VIII,Factor IX, tPA, hGH, receptors, insulin, vaccines, antibiotics and thelike. The coding sequences for proteins that can be used are known inthe art or canbe obtained by standard sequencing techniques.Alternatively, the vector may be used to produce an enzyme that is ableto convert a natural productto a unique product. This includes, forexample, the production of secondary metabolites useful aspharmaceuticals. Alternatively, the vectormay be used to producedegradative or inhibitory enzymes.

In an alternative embodiment, the vector is used for the transformationof crop plants to add some commercially desirable, agronomicallyimportant traits to the plant. Such traits include, but are not limitedto, herbicide resistance, increased yields, insect and diseaseresistance, physical appearance, food content and makeup, etc. Forexample, one may desire to incorporate one or more genes encodingherbicide resistance. Thebar and glyphosate tolerant EPSP synthase genesare good examples. A potential insect resistance gene which can beintroduced includes the Bacillus thuringiensis crystal toxin gene, whichmay provide resistance topests such as lepidopteran or coleopteran.

Genes encoding proteins characterized as having potential insecticidalactivity, such as the cowpea trypsin inhibitor (CpTI); may find use as arootworm deterrent; genes encoding avermectin may prove particularlyuseful as a corn rootworm deterrent. Furthermore, genes encoding lectinsmay confer insecticide properties (e.g., barley, wheat germ agglutinin,rice lectins) while others may confer antifungal properties (e.g.,hevein or chitinase).

The following examples illustrate the teachings of the present inventionand are not intended to limit the scope of the invention. In particular,although the following examples are limited to plant viruses and planthosts, the present invention also encompasses recombinant picorna-likeanimal virus vectors and the animal hosts injected by the animal virus.

Example 1

This example demonstrates the production of a potyvirus vectorcontaining aGUS marker, as also described in Dolja. V. V., et al., Proc.Nat'l Acad. Sci., 89:10208-10212 (1992), incorporated by referenceherein.

A DNA copy of TEV RNA (ATCC PV-69) was synthesized with reversetranscriptase (SuperScript from GIBCO-BRL) and a primer complementary to23 nucleotides (nt) preceding the 3'-terminal poly(A) tail of the virusgenome. Double-stranded DNA was generated using Escherichia coli DNApolymerase I and RNase H, digested with BstEII, and inserted intoBstEII-digested pTL7SN.3-0027, which contained sequences representingthe 5' and 3' ends of the TEV genome. A unique Bgl II site wasengineered immediately after the 25-nt 3' poly(A) tail by site-directedmutagenesis as described by Kunkel, T. A., et al., Methods Enzymol.,154:367-382 (1982), incorporated by reference herein. A longer poly(A)tail (70-75 nt)was incorporated from pBB5995, resulting in pTEV7D.Plasmids were propagated in E. coli strain HB101. FIG. 1 depictsdiagrams of portions ofplasmids containing cDNA representing thecomplete TEV genome and inserted GUS gene. The noncoding (open boxes)and coding (stippled shading) regionsof the TEV genome, the GUS gene(hatched shading), and SP6 RNA polymerase promoter (black arrow) areshown. Vertical lines below maps indicate sequences coding forproteolytic processing sites, whereas selected restriction sites areindicated above maps. Abbreviations as used in this and other Figuresare: N, Nco I; Bs, BstEII; Bg, Bg/II; 35, 35-kDa proteinase; HC, HC-Pro;50, 50-kDa TEV protein; CI, cylindrical inclusion protein; 6, 6-kDa TEVprotein; NIa, nuclear inclusion protein a; NIb, nuclear inclusionprotein b; Cap, capsid protein; 7D, pTEV7D.

Site-directed mutagenesis was used to introduce Nco I sites flanking the5'and 3' terminal coding sequences of the GUS gene in pTL7SN.3-GUS. TheGUS coding region was excised with Nco I and inserted into the Nco Isite thatwas introduced near the beginning of the HC-Pro coding sequencein pTL7SN.3-0627. The GUS gene and adjacent TEV coding sequences fromthis plasmid were incorporated into pTEV7D, resulting in pTEV7D-GUS.HC(FIG. 1). The GUS gene was also inserted into the Nco I site that hadbeen introduced previously at the beginning of the 35-kDa protein codingregionin pTL7SN.3-0027. The GUS and adjacent TEV sequences from theresulting plasmid were introduced into pTEV7D, yielding pTEV7D-GUS.P1(FIG. 1).

Transcripts that were capped with7-methylguanosine(5')triphospho(5')guanosine were synthesized usingbacteriophage SP6 RNA polymerase and cesium chloride-purified, BglII-linearized plasmid DNA as described by Carrington, J. C., et al., J.Virol., 64:1590-1597 (1990) incorporated by reference herein. The 5'ends of transcripts from pTEV7D and related plasmids contained twoadditional nonviral nucleotides. Transcription mixtures were dilutedwith equal volumes (vol) of 10 mM sodium phosphate buffer, pH 7.4, andapplied manually onto young tobacco plants (10 μl per leaf, two leavesper plant) with the aid of carborundum. In passage experiments, infectedleaves were ground in 10 vol of 10 mM sodium phosphate buffer containingcarborundum and applied manually to leaves of healthy plants with acottonswab.

Fluorometric assays for GUS activity and measurements of proteinconcentration were conducted as described by Carrington, J. C., et al.,J.Virol, 64:1590-1597 (1990), previously incorporated by referenceherein. Insitu GUS assays were done by using a colorimetric substrateaccording to Restrepo et al., Plant Cell, 2:987-998 (1990), incorporatedby reference herein. Tobacco leaves were vacuum infiltrated with thesubstrate 5-bromo-4-chloro-3-indolyl β-D-glucuronic acid,cyclohexylammonium salt (X-glue) (1.2 mM) in 0.5 mM potassiumferricyanide/10 mM EDTA. Manualcross-sections of leaf petioles, stems,and roots were placed directly intothe substrate solution andphotographed under bright-field optics by using a Zeiss photomicroscope.Total SDS-soluble proteins were extracted from leaf tissue by grindingin 7 vol of protein dissociation buffer (0.625M TrisoHCl, pH 6.8/2%SDS/10% 2-mercaptoethanol/10% (vol/vol) glycerol) and subjected toimmunoblot analysis with anti-HC-Pro, anti-capsid, or anti-GUS sera bydescribed procedures.

Approximately 27% (15 of 56) of tobacco plants inoculated with cappedRNA transcripts derived from pTEV7D became infected. Using the PCR, RNAisolated from progeny virions was shown to contain the sequencecorresponding to the Nco I site engineered at the start codon for TEVpolyprotein in pTEV7D, demonstrating the transcript-derived origin ofinfection. Twenty-five percent (16 of 64) and 0% (0 of 16) of plantsinoculated with pTEV7D-GUS.HC and pTEV7D-GUS.P1 transcripts,respectively,became infected. Due to the absence of infectivity,pTEV7D-GUS.P1 was not used further. Virion RNA from pTEV7D-GUS.HCtranscript-inoculated plants exhibited decreased electrophoreticmobility compared with RNA from wild-type TEV, suggesting that the GUSgene was retained in progeny virus.Plants infected by the modifiedvirus, which will be referred to as TEV-GUS, lacked the vein clearingand etching typical of plants infected by wild-type TEV.

The GUS gene in pTEV7D-GUS.HC was inserted adjacent to the codingsequence for the 35-kDa proteinase autoproteolytic cleavage site atTyr-304/Ser-305between the 35-kDa proteinase and HC-Pro. Insertion ofGUS into the polyprotein at this position has been shown not tointerfere with the 35-kDa proteinase processing activity. Proteolysis by35-kDa proteinase and HC-Pro at their respective C- termini waspredicted to yield a GUS-HC-Pro fusion protein of approximately 119 kDa.

FIG. 2 depicts the immunoblot analysis of extracts from RNAtranscript-inoculated tobacco plants. Extracts from pTEV7D (7D; lanes 6,10, and 14), two pTEV7D-GUS.HC (lanes 4, 5, 8, 9, 12, 13)-infectedplants,and a mock-inoculated plant (M; lanes 7, 11, and 15) wereanalyzed by usinganti-capsid protein, anti-HC-Pro, or anti-GUS serum.Lanes 4-15 each contained about 50 μg of SDS-soluble protein. A capsidprotein concentration series (capsid standard; Cap. std.) consisting of1.0 μg,0.1 μg, and 0.01 μg (lanes 1-3, respectively) was also analyzed.The molecular masses (in kDa) of capsid protein (30 kDa), HC-Pro (52kDa), andthe GUS-HC-Pro fusion protein (119 kDa) are located at left.The minor immunoreactive bands in lanes 8-20, 12, and 13 representdegradation products. Plants inoculated with transcripts from pTEV7D, onthe other hand, contained HC-Pro of normal size (52 kDa) that reactedonly with anti-HC-Pro serum (lanes 10 and 14). Immunoblot analysis usinganti-capsidserum indicated that the levels of capsid protein in plantsinoculated withpTEV7 D-GUS.HC and pTEV7D transcripts were similar (lanes4-6). By comparing the level of immunoreactive protein in preparationsof known protein concentration against a standard series, capsid proteinwas shown to account for about 1% of total protein in extracts fromplants inoculated with transcripts from either plasmid (lanes 1-6).Immunoblot analysis revealed accumulation of an approximately 119-kDaproduct that reacted with both anti-HC-Pro and anti-GUS sera (FIG. 2,lanes 8, 9, 12, and 13).

The stability of the GUS insert within the TEV-GUS genome was assayed byimmunoblot analysis in plant-to-plant passage experiments using virusoriginally recovered from two plants (plant 3 and 7) infected with RNAtranscripts. All passages were performed 4-6 days postinoculation(p.i.), except passage II (16 days p.i.). The 119-kDa GUS-HC-Pro proteinwas intact after seven passages of virus from plant 3 (FIG. 3, lanes 2,4, 6, 8, and 11), suggesting stable retention of the GUS sequence. InFIG. 3, samples of TEV-GUS were prepared from plants representing twoindependent virus lineages infected with pTEV7D-GUS.HC RNA transcriptsand were analyzed using anti-HC-Pro serum. Extracts from TEV-GUSpassages II-VII (lanes 1-11), pTEV7D transcript-inoculated (7D; lane12), and mock-inoculated (M; lane 13) plants are shown in FIG. 3. By useof a fluorometric assay, GUS activity levels were determined to becomparable in plants from each passage, as well as from an additionalseven passages.In contrast, TEV-GUS from plant 7 sustained a deletion inthe GUS-HC-Pro coding region that was evident during passage IV,resulting in appearance of an anti-HC-Pro reactive protein that wasabout 7 kDa smaller than intact HC-Pro (lanes 7, 9, and 10). This formwas also detected in the remaining three passages (lanes 1, 3, and 5).The disappearance of the GUS-HC-Pro protein correlated with a loss ofGUS activity, as well as a reversion to a mosaic- and etch-inducingphenotype, in plants from passages V-VII.

The stability of TEV-GUS as a function of time after inoculation wastestedby using virus from passage line 3. Upper systemic leaves frompassage V and VI plants were screened for GUS-HC-Pro fusion protein byimmunoblot analysis and for GUS activity by using a fluorometric assayat intervals after inoculation. FIG. 4 depicts analysis of GUS-HC-Profusion protein and GUS activity in aging plants infected by TEV-GUS. InFIG. 4(A), extracts were prepared at the times p.i. indicated from uppersystemic leaves of passage V and VI plants infected by TEV-GUS line 3and were subjected to immunoblot analysis with anti-HC-Pro serum (lanes1-6). Extracts were also analyzed from pTEV7D (7D; lane 7) andmock-inoculated (M; lane 8) plants. In FIG. 4(B), GUS activities asdetermined by fluorometric assay with extracts analyzed in panel A areshown. Deletions that resulted in truncated fusion proteins weredetected 20 and 25 days p.i. in passage VI and V plants, respectively(FIG. 4A, lanes 2 and 5, respectively). Additional deletion variantsencoding HC-Pro-related proteins smaller than wild-type HC-Pro wereevident after an additional 5 days in each plant (lanes 1, 4, and 7).The appearance of deleted forms correlated with decreased amounts ofboth the intact 119-kDa fusion protein (compare lanes 1 and 2 with lane3, and lanes 4 and 5 with lane 6)and GUS activity (FIG. 4B).

FIG. 5 depicts in situ localization of GUS activity in plants infectedby TEV-GUS. Plants were inoculated with TEV-GUS at 100 μg/ml. Wholeleaveswere vacuum-infiltrated with the histochemical GUS substrateX-gluc at the times p.i. (hr) indicated at bottom right. Panels A-C showmicroscopic, and panels D-F show macroscopic, visualization of GUSactivity in primary inoculated leaves. Note that panel B contains asingle GUS-positive epidermal cell. Panels G-I show microscopic andpanels J-L macroscopic visualization of GUS activity in upper systemicleaves. (Bars=200 μm.) Young tobacco plants were mechanically inoculatedwith TEV-GUS, and leaveswere excised and vacuum-infiltrated with GUSsubstrate X-gluc at several time points p.i. (FIG. 5). Single infectedepidermal cells that contained indigo GUS reaction product wereidentified 12 hr p.i. on primary inoculated leaves (panel B). By 24 hrp.i., movement to adjacent epidermaland mesophyll cells had resulted ininfection foci that extended to approximately 10 cells in diameter andthat were visible by eye (panels C and D). These foci continued toexpand and eventually fused by 96 hr p.i. (panels E and F). Measurementsof cell-to-cell movement over time indicated that focus expansionoccurred at a rate of approximately one cell per 2 hr. Activity ofTEV-GUS was detected around segments of vascular tissue in systemically(noninoculated) infected leaves by 60 hr p.i. (panel H). After 72 hrp.i., virus movement was evident along major and minor veins and intoleaf mesophyll cells adjacent to vascular tissue (panels I and K).Spread of virus through mesophyll tissue between veins proceed at a ratecomparable to that measured on inoculated leaves (panelsK and L).

To determine which cells or cell types were infected first duringsystemic spread, cross-sections from stems, roots, and leaf petiolesabove the siteof inoculation were incubated in X-gluc and visualized bylight microscopy.FIG. 6 depicts in situ localization of GUS activity incross-sections of petioles, stems, and roots of plants infected byTEV-GUS. Sections were cut by hand at the times p.i. (hr) indicated atthe bottom of panels A-L and incubated with the histochemical substrateX-gluc. Panels A-D show petioles; E-H depict stems; and I-L show roots.Abbreviations as used in this Figure are: C, cortex; LR, lateral root;P, phloem; and X, xylem. (Bars=200 μm.) No TEV-GUS activity was detectedat 24 and 48 hr p.i. inany of the organs (FIG. 6, A, B, E, F, I, and J).By 72 hr p.i., however, clusters of phloem-associated cells in each ofthe three organs exhibited activity (panels C, G, and K).Nonphloem-associated cell types, such as xylem parenchyma and cortex,were free of detectable TEV-GUS. Ingress intothese cell types wasevident after 96 hr p.i. (panels D, H, and L). Strikingly, tissues oflateral roots contained especially high levels of activity (panel L).

The TEV-GUS system demonstrated that potyviruses have utility asreplicating vectors for the introduction and expression of foreign genesin plants. For TEV, capsid protein accounts for about 1% of SDS-solubleprotein in infected leaves (FIG. 2). Unlike most other plant RNA virusvectors, the potyvirus-based system permitted both efficient systemicspread and high insertion capacity.

Example 2

This example shows the spontaneous mutagenesis of a plant potyvirusgenome after insertion of a foreign gene, as described in Dolja, V. V.et al., J.Virol., 67:5968-5875 (1993), incorporated by reference herein.

Routine mechanical inoculation of young Nicotiana tabacum cv. Xanthi ncplants with wild-type TEV-HAT (high aphid transmissibility and TEVmutantswas performed with the aid of carborundum using homogenatesprepared by grinding infected leaves in 2 vol of 10 mM Tris-HCl, 1 mMEDTA, pH 7.6. Homogenates were aliquoted, frozen at -85° C., and thawedprior to inoculation as needed. In some cases, crude virus preparationsfrom individual infected plants were obtained using the same method, butwith exclusion of the CsCl-gradient step. Viral RNA was purified usingproteinase K digestion, phenol extraction, and ethanol precipitation asdescribed by Carrington, J. C., et al., Virology, 139:22-31 (1984),incorporated by reference herein.

Complementary DNA spanning the region of deletion in the GUS-HC-Procoding sequence was obtained by reverse transcription of RNA from crudevirus preparations followed by PCR. Reverse transcription and PCR wereconductedaccording to established procedures as described by Kawasaki,E. S., In PCRProtocols, pp. 21-27 [Academic Press, Inc., San Diego(1990)], incorporatedby reference herein, except that reversetranscriptase buffer from Gibco/BRL was used, rather than PCR buffer inthe initial step. The first-strand primer was complementary to TEVnucleotides 1456-1477 within the HC-Pro coding region while thesecond-strand primer corresponded to nucleotides 781-799 within the P1region. The first and second-strand primers contained HindlII and BamHIrecognition sequences, respectively, to facilitate insertion into thevector pTL7SN. All plasmids were propagated in E. coli strain HB101. Thesequence of the insert DNA spanning the deletion endpoints inrecombinant plasmids was determined using the Sequenase® kit (U.S.Biochemicals) and a primer corresponding to TEV nucleotides 1015-1032.Each spontaneous deletion variant identified was assigned a code (1 del,2del, etc.).

The sequences between nucleotides 663 to 2416 in the genomes of deletionvariants TEV-2del and TEV-7del were reverse transcribed, amplified byPCR,and digested with SnaBI and EcoRI which recognize sites within theP1 and HC-Pro coding regions, respectively. The cleaved PCR fragmentswere clonedinto SnaBI and EcoRI-digested pTL7SN.3-0027DA, which containssequences corresponding to the 5'-terminal 2681 and 3'-terminal 154nucleotides of TEV genome. This plasmid was derived from pTL7SN.3-0027Dby removal of twononviral nucleotides separating the 5'-terminus of theTEV sequence from the start site of SP6 RNA polymerase transcription.The resulting plasmids, therefore, possessed the sequences surroundingthe deletion breakpoints that were present in TEV-2del and TEV-7del. Togenerate full-length copies of TEV genomes containing these deletions,the BstEII-BstEII fragment corresponding to TEV nucleotides 1430-9461was isolated from pTEV7D, and was inserted into BstEII-digestedderivatives ofpTL7SN.3-0027DA. FIG. 7 depicts genetic maps of TEV andselected TEV variants used in this study. FIG. 7(A) shows wild-typeTEV-HAT as derived from transcription of pTEV7DA. The single openreading frame is indicated by the extended, light gray box. Positionsencoding polyprotein cleavage sites are shown by the vertical lines. Thenames of individual proteins are shown above the map. FIG. 7(B) depictsTEV-GUS and deletion variants. TEV-GUS differs from TEV-HAT in that theformer contains the coding regionfor β-glucuronidase (GUS) insertedbetween the P1 and HC-Pro sequences. The polyprotein encoded by TEV-GUScontains a functional P1 cleavage site between P1 and GUS, resulting information of a GUS-HC-Pro fusion protein. The sequences missing in thedeletion variants of TEV-GUS are shown in the expanded diagram. Thenumbers indicate the nucleotide positions of the deletion endpoints inthe GUS and HC-Pro coding sequences. Abbreviations as used in this andother Figures are: HC-Pro, helper component-proteinase; CI, cylindricalinclusion protein; NIa, nuclear inclusion protein `a`; NIb, nuclearinclusion protein `b`; Cap, capsid protein. These plasmids were namedpTEV7DA-2del_(r) and pTEV7DA-7del_(r) (FIG. 7B). Viruses derived fromtranscripts of these plasmids were designated TEV-2del_(r) andTEV-7del_(r) (the `r` subscript indicates reconstructed virus). As acontrol, the same manipulations (reverse transcription, PCR andsubcloning) were performed with the wild-type TEV-HAT genome, resultingin plasmid pTEV7DA_(r).

The deletion mutant TEV-2del (and 2del_(r)) retained nucleotides 1-135from GUS but lost HC-Pro sequence up to nucleotide 207 (FIG. 7B). Twoadditional deletion variants were generated by site-directed "loop out"mutagenesis of pTEV7DA-2del_(r). The GUS sequence between nucleotides10-135 was removed, resulting in pTEV7DA-AGUS. The HC-Pro deletion inpTEV7DA-2del_(r) was enlarged by removal of sequence corresponding toHC-Pro nucleotides 208-265, yielding pTEV7DA-ΔHC (FIG. 7B).

RNA transcripts capped with m⁷ GpppG were synthesized usingbacteriophage SP6 RNA polymerase (Ambion) and CsCI gradient-purified,Bg/II-linearized plasmid DNA as described by Carrington, J. C., et al.,J.Virol., 64:1590-1597 (1990), previously incorporated by referenceherein. The undiluted transcription mixtures were applied manually ontoleaves of young tobacco plants (10 μl per leaf, two leaves per plant)that were dusted with carborundum.

Total SDS-soluble proteins were extracted from leaf tissue byhomogenization in 4 vol of protein dissociation buffer (0.625M Tris-HCl,pH 6.8, 2% SDS, 10% 2-mercaptoethanol, 10% glycerol) and subjected toimmunoblot analysis with anti-capsid or anti-HC-Pro sera. Quantitationof capsid protein levels in leaf extracts was done using capsid proteinstandards from purified virus and densitometry of immunoblots with aModel620 Video Densitometer (Bio-Rad).

Isolation of total RNA from leaf tissue was as described previously byCarrington, J. C., et al., Virology 139:22-31 (1984), previouslyincorporated by reference herein. RNA (5 μg) was denatured by glyoxaltreatment, separated by electrophoresis through a 1% agarose gel, andblotted onto a GeneScreenPlus™ membrane (DuPont). Northern hybridizationwas performed using a randomly-primed, ³² P-labeled probe prepared froma DNA fragment corresponding to TEV nucleotides 1430-9461. The amount ofradioactivity bound to each lane was measured using a Beckman LS 5801Scintillation Counter.

For transmission directly from infected leaves, aphids (Myzus persicaeSulz) were allowed a 5-10 min acquisition access period and thentransferred to tobacco (Nicotiana tabacum cv. Ky 14) seedlings. Tenaphids were placed on each of ten test plants for each treatment in eachexperiment. In helper component prefeeding experiments, aphids werefirst allowed a 5-10 min acquisition access period to a concentratedpreparationof potato virus Y helper component through a parafilmmembrane. Aphids werethen transferred to a TEV-infected leaf andprocessed as described above. Infection of test plants was scoredvisually.

As demonstrated previously in Example 1, the transfer of TEV-GUS fromplant-to-plant at intervals of four to six days resulted in stableretention of the GUS insert over 7 passages. Continuation of thisexperiment up to 21 passages during approximately 120 days furtherdemonstrated the stability of the foreign insert under these conditions.FIG. 8 depicts immunoblot analysis of extracts from upper leaves ofTEV-GUS-inoculated tobacco plants using anti-HC-Pro sera. Each lanecontains a sample from an individual plant inoculated with TEV-GUS,TEV-HAT, or buffer (lane M). All plants were propagated 4-5 weekspost-inoculation except the plant labeled TEV-GUS, which was propagatedfor 6 days post-inoculation. Each of the samples in lanes 1-12 containedaTEV-GUS-derived, spontaneous deletion variant that encoded a truncatedGUS-HC-Pro fusion protein. Lanes 4 and 3 contained extracts from plantsrepresenting first and second passages, respectively, that derived froma single TEV-GUS-infected plant. Each lane was loaded with equivalentamounts of extract. The weak appearance of HC-Pro-related proteins onlanes 1, 5, 7, and 12 was due to their low yield or rapid turnover. Themolecular weights in kilodaltons (kDa) of wild-type HC-Pro andGUS-HC-Pro fusion protein are shown in the Figure. Immunoblot analysisof total protein from the 21st passage plant revealed the full-size,GUS-HC-Pro fusion product (FIG. 8). The level of GUS activity in thisplant was comparable to that in plants from the early passages. Incontrast, prolonged propagation (3 to 4 weeks) of virtually all TEV-GUSinfected plants led to deletion mutants encoding HC-Pro-related proteinsthat were considerably smaller than GUS-HC-Pro (FIG. 8, lanes 1-12).Several of these variants, all of which were found in systemicallyinfected leaves, expressed HC-Pro forms that were smaller than thewild-type protein (52-kDa), indicating that these mutants containeddeletions of GUS and HC-Pro sequences. In some cases, apparentintermediate deletion routants were identified that, upon furtherpassage, were replaced by variants containing larger deletions (FIG. 8,compare lanes 4 and 3). In control plants infected for five weeks bywild-type TEV-HAT, no deletions of HC-Pro were evident (FIG. 8),suggesting that the deletion phenomenon was specific to TEV carrying theforeign gene.

Virions and corresponding RNA were purified from six individual plantsinfected by TEV-GUS deletion variants expressing HC-Pro-related proteinsof various sizes. The regions overlapping the deletion sites were copiedby reverse transcription, amplified by PCR, cloned in a plasmid, andsequenced. Analysis of 40 cDNA clones revealed 9 different deletionvariants (Table 2).

                  TABLE 2                                                         ______________________________________                                        Summary of TEV-GUS deletion mutants.                                                           Deletion 5'                                                                             Deletion 3'                                        Deletion                                                                             Deletion  end within                                                                              end within                                                                            Number of                                  Code.sup.a                                                                           length (nts)                                                                            GUS.sup.b HC-Pro.sup.c                                                                          cDNA clones.sup.d                          ______________________________________                                        1del   1787      31(G)     no deletion                                                                           5                                          2del   1890      135(T)    207(G)  1                                          3del   1828      80(A)      90(A)  1                                          4del   1939      66(T)     187(G)  9                                          5del   1741      92(A)      15(C)  3                                          6del   1789      81(T)      52(T)  2                                          7del   2074       9(T)     265(C)  10                                         8del   1804      91(G)      77(C)  8                                          9del   1840      84(G)     106(A)  1                                          ______________________________________                                         .sup.a Variants 4del, 5del, and 6del were isolated from the same plant, a    were variants 8del and 9del. All other variants were from individual           plants. Note: the numbering of deletions does not correspond to the           numbering of lanes in FIG. 8.                                                 .sup.b Numbering starts with the first nucleotide of the GUS coding           sequence. The last GUSderived nucleotide is shown in parentheses.             .sup.c Numbering starts with the first nucleotide of the HCPro coding         sequence, which corresponds to TEV genome nucleotide 1057. The first          HCPro-derived nucleotide is shown in parentheses.                             .sup.d Number of plasmids sequenced after cloning of PCR products.       

The deletions ranged from 1741 to 2074 nucleotides in length, showingalmost complete loss of the GUS gene in all mutants. Each mutant,however,retained a short segment of between 9 and 135 nucleotides fromthe 5' end of the GUS sequence. All but one of the variants also waslacking between 15 and 265 nucleotides from the 5' terminal region ofthe HC-Pro coding sequence. Interestingly, in no case were the 5' and 3'deletion endpoints entirely within the GUS coding region. All deletionsthat were mapped resulted in preservation of the open reading frame, andin some examples, led to the formation of new hybrid codons at thejunction sites. Two of the six plants analyzed contained multipledeletion mutants, although in both instances one type predominated amongthe population recovered (Table2). No mutants contained multipledeletions, nor were non-GUS or non-HC-Prosequences located at thejunctions, suggesting that the deletions were generated by removal ofcontiguous genome segments.

FIG. 9 depicts relative positions of deletion endpoints within the GUSand HC-Pro coding sequences in spontaneous mutants of TEV-GUS. Eacharrow indicates a unique endpoint identified by sequence analysis. Therelative positions of the 5' and 3' deletion endpoints are presenteddiagrammatically in FIG. 9. All of the 5' endpoints clustered within the130 nucleotide region of GUS gene, whereas the 3' endpoints all mappedto the first 265 nucleotides of the HC-Pro sequence. Alignment ofsequences flanking the endpoints of deletions failed to reveal anycommon elements of primary structure. In fact, the extent of homology ofsequences surrounding the junction sites did not exceed two nucleotidesin any of the mutants. Similarly, no nucleotide preferences at thedeletion endpoints were evident (Table 2).

The fact that the deletion mutants were able to spread systemicallysuggested that the HC-Pro region lacking in the mutants was notessential for virus replication and intraplant movement. Since it waspossible that these plants also contained a low level of nondeletedTEV-GUS providing functions necessary for these processes, uniform viruspopulations were prepared for the two variants (2del and 7del)containing the largest HC-Pro deletions. An approximately 350 nucleotidesegment covering the deletion site in both genomes was amplified by PCRand inserted into pTEV7DA. generating pTEV7DA-2del_(r) andpTEV7DA-7del_(r). The resulting constructs harbored the deletion in awild-type TEV-HAT background and lacked any other sequence changespotentially present in the original routants. As a control, thecorresponding genome segment fromwild-type TEV-HAT was amplified andcloned in pTEV7DA to produce pTEV7DA_(r).

Between 60 and 100% of plants inoculated with synthetic transcripts frompTEV7DA_(r), pTEV7DA-2del_(r) and pTEV7DA-7del_(r) became infectedsystemically, demonstrating conclusively that the mutants were capableof replication and movement independent of a nondeleted TEV-GUS. Theappearance of systemic symptoms at four days post-inoculation wasobservedin plants infected by TEV-HAT and the deletion mutants. Systemicsymptoms in plants inoculated with TEV-GUS, on the other hand, requiredfive days post-inoculation. FIG. 10 depicts immunoblot analysis withanti-HC-Pro serum of extracts from plants inoculated with synthetic RNAtranscripts. FIG. 10(A) depicts extracts were from systemically infectedleaves of three plants inoculated with transcripts from pTEV7DA-HAT_(r)(lanes 1-3) and three plants inoculated with transcripts frompTEV7DA-7del_(r) (lanes 4-6). FIG. 10(B) shows extracts from amock-inoculated plant (lane 1), plants inoculated with viruspreparations of wild-type TEV-HAT (lane 2) and TEV-2del (lane 6), orsynthetic RNA transcripts from pTEV7DA-2del_(r) (lane 3), pTEV7DA-AGUS(lane 4) and pTEV7DA-ΔHC (lane 5). Each lane was loaded with anequivalent amount of extract. The positions of HC-Pro and truncatedHC-Pro derivatives are shown at the leftof each panel. Immunoblotanalysis using anti-HC-Pro serum indicated that the two reconstructeddeletion variants encoded truncated HC-Pro-related proteins, whereas thereconstructed wild-type virus expressed a normal size product (FIG. 10Aand 10B, lane 3).

Although the N-terminal HC-Pro sequences clearly were not essential forvirus viability, it was noticed that the levels of HC-Pro appeared to beconsiderably less in plants infected by the mutants compared towild-type TEV-HAT. Also, the level of GUS-HC-Pro fusion protein inTEV-GUS-infected plants appeared less than HC-Pro in plants infected byTEV-HAT. To determine more precisely the levels of replication ofwild-type and mutantviruses in systemically infected tissue, therelative amounts of capsid protein and virus RNA were measured usingimmunoblot and Northern blot analyses, respectively.

FIG. 11 depicts quantitation of TEV capsid protein (panels A and B) andRNA(panels C and D) present in systemically infected leaves of plantsinoculated with wild-type and mutant viruses. Panels 11(A) and 11(B)show capsid protein levels in total protein extracts were determined byreflective densitometry of immunoblots using capsid protein standardsfrompurified virions. Each bar represents the mean (and standarddeviation) of three independent samples. Panels 11(C) and 11(D) depictrelative viral RNA levels in total RNA extracts determined by measuringradioactivity from a ³² P-labeled probe bound to Northern blots. Twoindependent samples are shown in each case. For comparative purposes,note that data shown in panels A and C were collected fromcontemporaneous samples in oneexperiment, while data in panels B and Dwere from another set of contemporaneous samples. The insertion of GUSinto the viral genome in TEV-GUS decreased the yield of capsid proteinand RNA to levels of 15 and 9%, respectively, compared to wild-type(FIG. 11A and C). The presence of GUS, therefore, had a debilitatingeffect on accumulation of virus. TEV-7del, which specified three aminoacid residues from GUS but possessedan HC-Pro deletion to residue 89,yielded even less viral protein and RNA at 8 and 6% of the wild-typelevels, respectively (FIG. 11A and C). In contrast, plants infected byTEV-2del, which coded for 45 residues of GUS but lacked 66 HC-Proresidues, accumulated capsid protein and RNA at 25 and 17% of thewild-type levels, respectively (FIG. 11A and C). TEV-2del, therefore,replicated to a degree approximately three times higher than TEV-7del.

The differences between the two spontaneous mutants may have been due tothe presence of the short GUS sequence having a stabilizing effect inTEV-2del_(r) or to the larger deletion within TEV-7del affecting acritical function of HC-Pro. To distinguish between these twopossibilities, two site-directed mutations were introduced intopTEV7DA-2del_(r). In the first, pTEV7DA-ΔGUS, the GUS sequence betweennucleotides 10-135 was deleted, leaving a short GUS region equivalent tothat present in TEV-7del. In the second, pTEV7DA-ΔHC, the HC-Prodeletion was extended from nucleotide 207 to 265, correspondingto thesequence missing in TEV-7del. Immunoblot analysis with HC-Pro-specificantibodies revealed the truncated HC-Pro-related products of theexpected sizes in plants inoculated with transcripts from the twomutants (FIG. 10B, lanes 4 and 5). Based on the apparent quantities ofHC-Pro deletion products in these plants, TEV-ΔGUS replicated betterthanTEV-2del_(r) and TEV-ΔHC. This was confirmed by quantitation of thelevels of capsid protein and RNA in systemic leaves. Compared toTEV-2del_(r), TEV-ΔGUS directed an average of approximately 70% morecapsid protein and RNA, while TEV-ΔHC directed less of these products(FIG. 11B and D). This demonstrated that the presence of the GUSfragment, and the absence of an additional HC-Pro sequence, bothconferreda debilitating effect on virus accumulation.

The ability of aphids to transmit TEV-HAT, TEV-GUS, TEV-2del andTEV-7del was tested. Transmission assays demonstrated that only TEV-HATpossessed an aphid-transmissible phenotype (Table 3).

                  TABLE 3                                                         ______________________________________                                        Aphid transmission of wild-type and mutant TEV variants.                                  Without HC-Pro                                                                             With HC-Pro                                          Virus       prefeeding   prefeeding.sup.a                                     ______________________________________                                        TEV-HAT     34/40.sup.b  10/10                                                TEV-GUS     0/40         0/20                                                 TEV-2del    0/30         4/30                                                 TEV-7del    0/30         0/20                                                 ______________________________________                                         .sup.a Aphids were allowed access to active HCPro from potato virus Y         prior to transmission assays.                                                 .sup.b Data are shown for infected plants/total plants assayed.          

The lack of transmission of TEV-GUS, TEV-2del and TEV-7del could havebeen due to functional defects in the mutant HC-Pro proteins, and/or tothe decrease in mutant virus titers and corresponding low concentrationsof HC-Pro. Prefeeding of aphids on concentrated preparations of HC-Procan facilitate transmission of mutant viruses expressing defectiveHC-Pro proteins. Using highly active preparations of potato virus YHC-Pro, aphidtransmission of TEV-2del was restored, although with lowefficiency (Table 3). The inability to transmit TEV-GUS or TEV-7del withthe aid of prefeeding was due most likely to low levels of virusaccumulation (FIG. 11A, C).

Example 3

This is a prophetic example. The gene encoding the sequence for humaninsulin is inserted into a TEV vector as described for GUS in Example 1.The expression vector is applied manually to plants as in Example 1 toproduce human insulin. Human insulin is extracted from the plants.

Many other variations and modifications may be made in the methodsherein described by those having experience in this art, withoutdeparting from the concept of the present invention. Accordingly, itshould be clearly understood that the methods described in the foregoingdescription are illustrative only, and not intended as a limitation onthe scope of the invention.

What is claimed is:
 1. A plasmid comprising:(a) at least one promoter;(b) cDNA, wherein said cDNA comprises sequences that code for areplicatable genome of a polyprotein-producing Tabacco Etch Virus, and;(c) at least one unique restriction site flanking a 3' terminus of saidcDNA.
 2. A plasmid comprising:(a) at least one promoter; (b) cDNA,wherein said cDNA comprises sequences that code for a replicatablegenome of a polyprotein-producing Tobacco Etch Virus, and wherein saidpolyprotein comprises at least one protein non-native to the TobaccoEtch Virus, and; (c) at least one unique restriction site flanking a 3'terminus of said cDNA.
 3. The plasmid according to claim 2 wherein saidcDNA sequence for said polyprotein has a coding sequence for saidprotein non-native to the Tobacco Etch Virus inserted between codingsequences for proteins native to said polyprotein-producing Tobacco EtchVirus.
 4. A method for producing a virus, said method comprising:(a)reverse transcribing a polyprotein-producing Tobacco Etch Virus RNAgenome into cDNA; (b) introducing at least one unique restriction siteflanking a 3' terminus of said cDNA; (c) inserting said cDNA into acloning vehicle; (d) producing RNA transcripts from said cDNA using anRNA polymerase; and (e) inoculating plants or plant cells with said RNAtranscripts.
 5. A method for producing a virus, said methodcomprising:(a) reverse transcribing a polyprotein-producing tobacco EtchVirus RNA genome into a first cDNA; (b) introducing at least one uniquerestriction site flanking a 3' terminus of said first cDNA; (c)inserting into said first cDNA a second cDNA sequence wherein saidsecond cDNA sequence codes for a protein non-native to the Tobacco EtchVirus; (d) inserting said first and second cDNA into a cloning vehicle;(e) producing RNA transcripts from said cDNA using an RNA polymerase;and (f) inoculating plants or plant cells with said RNA transcripts. 6.A method for expressing at least one protein in a plant or plant cell,said method comprising infecting a plant or plant cell susceptible to apolyprotein-producing Tobacco Etch Virus with said Tobacco Etch Virus,expressing said Tobacco Etch Virus to produce said polyprotein, whereinsaid Tobacco Etch Virus codes for at least one protein non-native to theTobacco Etch Virus and wherein said non-native protein is released fromsaid polyprotein by proteolytic processing.
 7. The method according toclaim 6 wherein said protein non-native to the Tobacco Etch Virus is apharmaceutical.
 8. The method according to claim 6 wherein said proteinnon-native to the Tobacco Etch Virus is selected from the groupconsisting of insulin, hGH, interleukin, EPO, G-CSF, GM-CSF. hPG-CSF,M-CSF, Factor VIII, Factor IX, and tPA.
 9. The method according to claim6 wherein said protein non-native to the Tobacco Etch Virusmetabolically interacts with a compound that occurs in the host cell toproduce a secondary metabolite of said compound.